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Endocrinology, doi:10.1210/en.2006-0579
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Endocrinology Vol. 147, No. 12 5835-5844
Copyright © 2006 by The Endocrine Society

Sphingosine-1-Phosphate Modulates Both Lipolysis and Leptin Production in Differentiated Rat White Adipocytes

Dong-Jae Jun, Jong-Hee Lee, Bo-Hwa Choi, Tae-Kyung Koh, Dae-Cheong Ha, Min-Woo Jeong and Kyong-Tai Kim

Systems Bio-dynamics National Core Research Center, Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang 790-784, Korea

Address all correspondence and requests for reprints to: Kyong-Tai Kim, Ph.D., Department of Life Science, POSTECH, San 31, Hyoja Dong, Pohang 790-784, Korea. E-mail: ktk{at}postech.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Sphingosine-1-phosphate (S1P) is a pluripotent lipid mediator that transmits signals through a family of G protein-coupled receptors to control diverse biological processes. Here, we investigated the effects of S1P on the levels of intracellular calcium and cAMP in differentiated rat white adipocytes and two important aspects of adipocyte-specific physiology, lipolysis and leptin production. In adipocytes, S1P signaling pathway was functionally linked to phospholipase C via pertussis-toxin-sensitive G protein. Interestingly, at higher S1P concentration (1–30 µM), it also induced cAMP generation in a concentration-dependent manner, which was pertussis toxin insensitive and was mimicked by dihydro-S1P and sphingosylphosphoryl-choline but not by its related metabolites, ceramide and sphingosine, or by its structural analogs, phyto-S1P and lysophosphatidic acid. Suramin, a known inhibitor of ligand-receptor interactions, reduced S1P-induced cAMP generation by 60% of control, whereas forskolin-induced cAMP increase was not affected by treatment with suramin. The S1P-induced cAMP generation was functionally linked to cAMP response element-binding protein phosphorylation. Finally, S1P significantly reduced insulin-induced mRNA of ob gene and leptin secretion, whereas S1P increased glycerol release from adipocytes. Both effects of S1P were reversed by a selective adenylyl cyclase inhibitor, SQ22536, without significantly affecting basal values. In conclusion, extracellular S1P elicits the elevation of cytosolic Ca2+ and cAMP with a distinct concentration dependency, and S1P-induced cAMP generation may be mediated by S1P-selective receptors rather than intracellular targets, and the activated adenylyl cyclase-cAMP signaling pathways subsequently increase lipolysis and decrease insulin-induced leptin production in rat white adipocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPHINGOSINE-1-PHOSPHATE (S1P), A bioactive lipid produced from the metabolism of sphingolipids has essential roles in many aspects of cell biology, from inflammatory responses through cell proliferation and apoptosis to cell migration and differentiation, and may affect pathophysiological diseases, such as atherosclerosis, autoimmune disease, and cancer (1, 2). S1P is stored in high concentrations and is released from human platelets upon activation by physiological stimuli but can also be synthesized in a wide variety of cell types in response to extracellular stimuli, such as growth factors and cytokines (3). These newly generated and secreted S1P are bound extensively by albumin and other plasma proteins that provide both a stable reservoir in extracellular fluids, resulting in higher total concentrations than in tissues, and efficient delivery to high-affinity cell surface receptors (4). Although S1P is taken up by the intracellular compartments and acts as a second messenger because of its lipophilic nature, it exerts most of its effects by binding to five related G-protein-coupled receptors in the plasma membrane, termed S1P1–5, possessing distinct expression profiles and affinities toward S1P (5). Each receptor signals to multiple downstream responses by coupling to different G proteins. S1P1 exclusively links to Gi; S1P2,3 links to Gi, Gq, G12/13; and S1P4,5 links to Gi and Gq (6) and thereby specifically control adenylyl cyclase, phospholipases C and D (PLC and PLD), phosphatidyl inositol-3-kinase (PI3K), ERK, c-Jun N-terminal kinase (JNK), p38, small GTPases such as Rac and Rho and probably nonreceptor protein tyrosine kinases and tyrosine phosphatases (7). This endows S1P with the ability to regulate diverse physiological processes in many organ systems. However, the study of physiological roles of S1P in white adipocytes is lacking.

Lipolysis is the hydrolysis of the ester bonds in triacylglycerol, which is composed of three fatty acids esterified to glycerol. Dysregulation of the lipolytic pathway has been one of the major hypotheses linking insulin resistance to hyperlipidemia in metabolic diseases, obesity, and diabetes mellitus (8, 9). Recently, the novel paradigm of adipose tissue as an endocrine organ was developed. Adipose tissue secretes a variety of endocrine hormones such as leptin, IL-6, angiotensin II, adiponectin, and resistin (10). From this viewpoint, adipose tissue plays a critical role as an endocrine gland, secreting numerous factors with potent effects on the metabolism of distant tissues. Leptin is a 16-kDa hydrophilic protein mainly secreted from white adipocytes, which is considered to signal the size of adipose tissue stores to the brain and plays a key role in regulating body weight (10, 11). Significant insight into leptin-induced regulation of metabolism has been achieved. However, the signaling pathway that regulates leptin production and secretion from the adipose tissue is still incompletely understood. Therefore, understanding the regulation of two important adipocyte-specific physiological processes, lipolysis and leptin production, is very important. Here we report that extracellular S1P elicits the elevation of cytosolic Ca2+ and cAMP with a distinct concentration dependency in rat white adipocytes, and the activated cAMP-protein kinase A signaling pathway subsequently increases lipolysis and decreases insulin-induced leptin production.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Fibroblastic preadipocytes were isolated from adipose tissue according to a previously reported method (12). Briefly, the sc fat pads from 14-d-old male Sprague Dawley rats (10 per group) were transferred in a buffer containing collagenase type II (1 mg/ml; Sigma Chemical Co., St. Louis, MO). After incubation for 1 h at 37 C in a shaking water bath, the digest was filtered through sterile 250 mm nylon mesh and was centrifuged at 2500g for 10 min. The pellet was resuspended in medium 199 (BioWhittaker, Walkersville, MD), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), penicillin (100 kU/liter) and streptomycin (100 mg/liter) (GIBCO BRL, Gaithersburg, MD), and refiltered. The cell preparation was adjusted to a density of 1.5 x 105 cells/ml. After 2 ds in culture at 37 C in an atmosphere of 5% CO2, differentiation was induced by the addition of medium supplemented with isobutylmethylxanthine, dexamethasone, and insulin (0.5 mM, 0.25 mM, and 10 mg/ml, respectively; Sigma). After 48 h, the induction medium was removed and replaced by medium 199 containing 10% fetal bovine serum supplemented with insulin (10 mg/ml) alone. This medium was changed every 2 d.

RT-PCR analysis
Total RNA was extracted from the adipocytes by TRI reagent (Molecular Research Center, Cincinnati, OH), and 1 µg of total RNA was reverse-transcribed with the use of SuperScript II reverse transcriptase (GIBCO BRL, Life Technologies). cDNA was amplified with 20 pmol of specific oligonucleotide primers (Bioneer, Daejon, Korea) using Ex Taq polymerase (Takara, Ohtsu, Japan). Primer sequences for the five subtypes and the reaction conditions were used as reported previously (13). The PCR products were analyzed on a 1% agarose gel and by sequencing.

Western blot analysis
Adipocytes were plated in 60-mm tissue culture dishes and treated with S1P for various times. After treatment, the cells were washed twice with cold PBS and then lysed with lysis buffer [250 mM Tris-Cl (pH 6.5), 2% SDS, 4% ß-mercaptoethanol, 0.02% bromophenol blue, and 10% glycerol]. Equal amounts of whole-cell lysates were resolved by 12.5% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Membranes were blocked using Tris-buffered saline with Tween 20 (150 mM NaCl, 10 mM Tris-HCl, pH 8.0, and 0.05% Tween 20) containing 5% skim milk for 30 min and then incubated overnight with the indicated primary antibody. After washing three times with Tris-buffered saline with Tween 20, the membranes were probed with horseradish-peroxidase-conjugated secondary antibody to allow detection of the appropriate bands using an ECL detection system (Neuronex Co., Pohang, Korea).

Confocal microscopy
To record fluorescence images, adipocytes cultured on poly-D-lysine-coated cover slips were preloaded with 5 µM fluo-4/AM dye. After incubation for 30 min at 37 C, the cells were washed two times in Locke’s solution to remove excess dye and examined under the confocal microscope. Measurements of intracellular calcium were performed with the Bio-Rad Radiance 2100 confocal microscope (Bio-Rad, Inc., Hemel Hempstead, UK) equipped with a x40 objective with 0.75 numerical aperture). The calcium-sensitive fluo-4 dye was excited by the 488-nm line from an argon laser, and the emission fluorescence monitored at 515 ± 15 nm was selected by a band-pass filter. During fluorescence data collection, each scan of a 512- x 512-pixel image took 0.35 sec, and the interval between each image scan was approximately 2 sec. Images were stored and processed with laser pix software (Bio-Rad). The regions of interest distributed across the image provided an intensity vs. time graphic output.

Measurement of intracellular Ca2+ level
Intracellular Ca2+concentration ([Ca2+]i) was determined using the fluorescent Ca2+ indicator fura-2 as previously reported (12). Briefly, adipocytes were loaded with fura-2 pentaacetoxymethyl ester (fura-2/AM) to a final concentration of 3 µM in complete medium and incubated at 37 C with stirring for 50 min. After the loading, fluorescence ratios were taken by dual excitation at 340 and 380 nm and emission at 500 nm with an alterative wavelength time scanning method. Calibration of the fluorescence signal in terms of [Ca2+]i was performed according to Grynkiewicz et al. (14).

Quantification of inositol-1,4,5-trisphosphate (IP3)
Concentration of IP3 in cells was determined by competition assay with [3H]IP3 as we described in detail elsewhere (15).

Measurement of [3H]cAMP
Intracellular cAMP generation was determined by [3H]cAMP competition assay in binding to cAMP-binding protein as described previously by Suh et al. (15) with some modifications. To determine the cAMP production induced by sphingosine 1-phosphate, the adipocytes were stimulated with agonists for 20 min in the presence of the phosphodiesterase inhibitor Ro 20-1724 (5 µM), and the reaction was quickly terminated by three repeated cycles of freezing and thawing. The samples were then centrifuged at 12,000 x g for 5 min at 4 C. The cAMP assay is based on the competition between 3H-labeled cAMP and unlabeled cAMP present in the sample for binding to a crude cAMP-binding protein prepared from bovine adrenal cortex according to the method of Brown et al. (16). Bound [3H]cAMP in the supernatant was then determined by liquid scintillation counting. Each sample was incubated with 50 µl 3H-labeled cAMP (5 µCi) and 100 µl binding protein for 2 h at 4 C. Separation of protein-bound cAMP from unbound cAMP was achieved by absorption of free cAMP onto charcoal (100 µl), followed by centrifugation at 12,000 x g at 4 C. The 200 µl supernatant was then placed into an Eppendorf tube containing 1.2 ml scintillation cocktail to measure radioactivity. The cAMP concentration in the sample was determined based on a standard curve and expressed as picomoles per microgram of protein.

Detection of leptin secretion
By using cultured rat adipocytes grown in 12-well plates, we measured the influence of various concentration of S1P on leptin production. Leptin concentration in the medium was determined with a sensitive and specific rat leptin RIA kit (Linco Inc., St. Charles, MO; catalog no. RL-83K).

Lipolysis measurements
Glycerol release was measured as previously described (17). Briefly, the differentiated adipocytes within 24-well plate were stimulated with agents for 24 h. After the stimulation, 1 ml hydrazine buffer containing 50 mM glycine (pH 9.8), 0.05% hydrazine hydrate, and 1 mM MgCl2 supplemented with 0.75 mg/ml ATP, 0.375 mg/ml nicotinamide adenine dinucleotide, 25 µg/ml glycerol-3-phosphate dehydrogenase, and 0.5 µg/ml glycerokinase was added to 200 µl collected media. After incubation for 40 min in room temperature, OD 340 was measured and glycerol release calculated.

Statistical analysis
All numerical values are given as mean ± SD. Significance of differences between mean values of two groups was evaluated using Student’s t test for paired or unpaired data as appropriate. A probability of P < 0.01 or P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Multiple subtype expression of S1P receptors in both differentiated rat white adipocytes and preadipocytes
S1p1, S1p2, S1p3, S1p4, and S1p5 are G-protein-coupled receptors that are specifically activated by S1P (18, 19, 20, 21, 22, 23, 24). To examine expression of the five known S1P receptor genes in differentiated rat white adipocytes, we performed RT-PCR with primers designed to specifically amplify a fragment of the rat S1P receptor cDNAs (13). As shown in Fig. 1Go, preadipocytes and differentiated rat white adipocytes expressed mRNA for S1p1, S1p2, S1p3, and S1p5 receptors, as indicated by the presence of bands of 273, 720, 460, and 300 bp, respectively. Nucleotide sequence analysis confirmed that the amplified DNA products of adipocytes were authentic rat S1p1, S1p2, S1p3, and S1p5 receptors. However, transcripts for S1p4 receptor could not be detected in adipocytes. This result was well correlated with previous reports that S1p4 expression was not detected in the adipose tissue, and in contrast to the other S1P receptor, the S1p4 expression profile was largely confined to the tissue and cells of the hematopoietic system (25, 26).


Figure 1
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  		FIG. 1. RT-PCR analysis detects the presence of mRNAs for S1p1, S1p2, S1p3, and S1p5 receptors. RT-PCR was performed from 1 µg total RNA extracted from rat white adipocytes and preadipocytes. Thirty-five PCR cycles were conducted from the same RT with specific primers for S1p1–5(see Materials and Methods). The products were analyzed by agarose-gel electrophoresis. Lane SM, DNA standards; lane Pre, preadipocytes; lane Dif, differentiated adipocytes; lane L, lung (positive control for S1p4).

 
Characterization of S1P-induced Ca2+ and IP3 changes in differentiated rat white adipocytes
There are a number of studies in which S1P has been shown to activate PLC-catalyzed inositol phosphate formation and subsequent calcium mobilization via Gq/11 or Gi/o pathways (27, 28, 29, 30, 31). To characterize S1P signal transduction pathway in rat white adipocytes, we therefore first investigated the effects of S1P on intracellular Ca2+ increase and phosphatidylinositol 4,5-bisphosphate hydrolysis. Stimulation of adipocytes with 1 µM S1P in the presence of 2.2 mM CaCl2 caused a transient elevation of the [Ca2+]i (Fig. 2AGo). The [Ca2+]i peaked within 15 sec after stimulation and then declined to the basal level within 2 min. In the absence of extracellular Ca2+, the S1P-induced Ca2+ mobilization was reduced by approximately 50% (Fig. 2AGo), suggesting that the increase in the [Ca2+]i was caused not only by the mobilization of Ca2+ from intracellular stores but also by influx of Ca2+ from the extracellular medium. We performed a similar analysis with regard to the effect of the S1P on IP3 generation. Stimulation of the cells with 1 µM S1P resulted in IP3 generation with the peak obtained within 15 sec after stimulation, and the effect was returned to basal level after 2 min (Fig. 2BGo). Because during terminal differentiation, fibroblastic preadipocytes divide and differentiate but a fibroblast layer can remain, we performed single-cell calcium imaging to confirm Ca2+ response of S1P in differentiated adipocytes with a confocal microscope. The addition of S1P to differentiated adipocytes elicits a typical transient increase in [Ca2+]i characterized by a rapid increase in [Ca2+]i followed by a slower decrease (Fig. 2CGo, h). In addition, Fig. 2CGo, a–d) illustrated intracellular spatial heterogeneity of the responses in differentiated adipocytes to 1 µM S1P by showing a highly localized rise in intracellular calcium in the perinuclear region of the cell. The three-dimensional surface plots of images represented more clearly the intracellular heterogeneity of calcium increase in individual adipocytes (Fig. 2CGo, e and f). This intracellular spatial variability in cytoplasmic [Ca2+]i was consistent with the report that there are clearly hot spots in which [Ca2+]i is much higher than either adjacent or distant areas (32), and it could be attributed to the unique geometry of the cell, which has the greater cytoplasmic volume near the nucleus compared with the lipid droplet-enriched cytoplasm. The addition of S1P to preadipocytes elicit a calcium increase pattern similar to that of differentiated adipocytes (Fig. 2DGo). These results demonstrated that preadipocytes and differentiated adipocytes express S1P receptor, which are functionally linked to PLC, to cause IP3 production and subsequent Ca2+ elevation.


Figure 2
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  		FIG. 2. S1P induces intracellular Ca2+ increase and IP3 generation in rat white adipocytes. A, Fura-2/AM-loaded adipocytes were challenged with 1 µM S1P with (left) or without (right) 2.2 mM extracellular CaCl2, and typical Ca2+ transients are presented. B, Adipocytes were stimulated with 1 µM S1P for the designated times (0, 15 sec, 30 sec, 3 min, 5 min, and 10 min), and the IP3 production was measured by competition assay as described under Materials and Methods. The experiments were done three times, and each point is the mean ± SD. *, P < 0.05. C and D, For intracellular calcium imaging in differentiated adipocytes (C) and preadipocytes (D), cells were loaded with fluo-4 as described in the Materials and Methods; a–d, fluo-4 fluorescence images of cells obtained at four different times after 1 µM S1P treatment (each image was obtained at that time indicated in h). The color scale indicates higher calcium concentrations in areas of yellow and red, whereas lower calcium concentrations are designated with light and dark blue. g, DIC image; h, detailed kinetics of calcium responses in the regions of interest (ROI) expressed as relative intensity of fluorescence (y-axis) vs. time (x-axis), and S1P was treated for the duration indicated by the horizontal bar; e and f, surface plots corresponding to the intensity of images a and b, respectively.

 
Adipocytes showed distinct concentration profiles between Ca2+ increase and cAMP generation upon S1P stimulation
To test the involvement of S1P receptor in the activation of adenylyl cyclase, we stimulated adipocytes with various concentrations of S1P. Interestingly, intracellular cAMP generation became apparent at 3 µM S1P and continuously increased. The concentration-response curve of S1P on cAMP generation showed a more right-shifted pattern than that on Ca2+ increase. EC50 values for S1P were approximately 27 nM for Ca2+ and 29 µM for cAMP, respectively (Fig. 3AGo). To determine the functional significance of S1P-induced cAMP generation in adipocytes, we tested cAMP response element-binding protein (CREB) phosphorylation as downstream of the adenylyl cyclase-cAMP pathway. In Fig. 3BGo, treatment of cells with 30 µM S1P led to the time-dependent phosphorylation of CREB; maximal stimulation occurred at 15 min, which then returned to basal level at 1 h. In contrast with 30 µM S1P, 500 nM S1P did not induce phosphorylation of CREB, whereas the phosphorylation of MAPKs, including JNK (Fig. 3BGo), ERK, and p38 (data not shown), were observed, and the phosphorylation patterns were not different between two concentrations of S1P. These results showed that S1P stimulated adenylyl cyclase at distinct concentrations from that of calcium increase and that S1P-induced cAMP generation was functionally linked to CREB phosphorylation.


Figure 3
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  		FIG. 3. Dose profile of S1P on cAMP generation was distinct from that of Ca2+ and was not affected by PTX pretreatment or Ca2+ chelation. A, Dose-response curves of S1P on Ca2+ increase and cAMP generation. Adipocytes were stimulated with various concentration of S1P, and the peak of internal Ca2+ levels and internal cAMP contents were measured as described in Materials and Methods. B, Comparison of CREB and JNK phosphorylation between 500 nM and 30 µM S1P-treated adipocytes. Whole-cell extracts from adipocytes treated with 500 nM or 30 µM S1P for various times were analyzed by Western blot of phospho-CREB or phospho-JNK. Equal loading of protein was checked by CREB, JNK, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. C and D, Effects of PTX pretreatment on S1P-induced Ca2+ increase and cAMP generation. Adipocytes were pretreated with vehicle or PTX (100 ng/ml for 18 h) and then stimulated with various concentrations of S1P. E, Effects of Ca2+ chelation on S1P-stimulated cAMP generation. BAPTA/AM (100 µM) and EGTA (300 µM) were loaded for 30 min and 1 min before stimulation with 30 µM S1P. cAMP contents was measured as described in Materials and Methods. All experiments were done three times, and each point is the mean ± SD.

 
Lack of relationship between S1P-induced Ca2+ increase and cAMP production
A number of studies have shown that S1P receptors could couple to mainly Gi protein in a pertussis toxin (PTX)-sensitive pathway (27, 28, 29, 30, 31). cAMP synthesis can be mediated by cross-talk with the Gi/PLC signaling pathways through Ca2+/calmodulin-sensitive or protein-kinase-C-stimulated adenylyl cyclase isoforms (6). To address whether PTX-sensitive G proteins are involved in the regulation of adenylyl cyclase activation, adipocytes were pretreated with PTX (100 ng/ml for 18 h), which ADP ribosylates and inactivates the {alpha}-subunits of Gi/Go and thereby maintains Gi/Go in a nondissociated form. However, S1P-induced cAMP generation was unaffected by PTX preincubation, whereas S1P-induced Ca2+ increases were strongly inhibited with more than 85% of untreated control (Fig. 3Go, C and D). In addition, even when adipocytes incubated with intracellular and extracellular Ca2+ chelators BAPTA/AM (100 µM) and EGTA (300 µM), respectively, in conditions that completely blocked the intracellular Ca2+ increase by 1 µM S1P treatment (data not shown), the S1P-induced elevation of cAMP was not affected. These results indicated that S1P-induced cAMP generation was not mediated by PTX-sensitive G proteisn and was unrelated to downstream signaling pathways of PLC/Ca2+ (Fig. 3EGo)

Action of S1P on adenylyl cyclase through S1P-specific receptor rather than its metabolic conversion or intracellular targeting
S1P is formed through the phosphorylation of sphingosine, catalyzed by sphingosine kinase (33). Cells also contain S1P phosphohydrolase and ceramide synthase activities, allowing S1P to be converted back to ceramide (34, 35). Indeed, cells maintain a dynamic equilibrium between the levels of ceramide, sphingosine, and S1P, and their relative levels determine the physiological fate of the cell (3). However, sphingosine and ceramide did not elevate basal cAMP level even at 50 µM, arguing against a secondary effect of S1P related to its metabolite conversion (Fig. 4AGo). Although direct intracellular targets for S1P have yet to be identified, a high concentration of S1P could act as an intracellular second messenger because of its lipophilic nature (36). To determine whether S1P stimulates cAMP generation via receptor or intracellular action, we used dihydro-S1P (Dh-S1P), which is identical to S1P and lacks only the trans 4,5 double bond, binds to all of the S1P receptors and activates them, yet does not mimic all of the intracellular effects of S1P (36, 37). As shown in Fig. 4BGo, when differentiated adipocytes were treated with various concentrations of Dh-S1P, the intracellular cAMP level was increased in a concentration-dependent manner comparable to the response of S1P. In addition, another structurally related analog, sphingosylphosphoryl-choline (SPC), also stimulated cAMP generation, albeit with reduced potency and efficacy. These agonisms of Dh-S1P and SPC in differentiated adipocytes was well correlated with activation of many known S1P receptors, which were S1p1/endothelial cell differentiation gene (EDG)-1, S1p3/EDG-3, S1p2/EDG-5, and S1p5/EDG-8 receptors expressed in Xenopus oocytes or in HEK 293T cells, with one exception that all of the effective concentrations of S1P, Dh-S1P, and SPC in the known S1P receptors were in the nanomolar range (20, 38). Phyto-S1P (Pt-S1P) was reported to bind to S1p4 receptor with higher affinity than S1P (39). However, in adipocytes, Pt-S1P did not affect basal cAMP level even with its concentration up to 100 µM, and thereby the involvement of S1p4 receptor in S1P-induced cAMP generation could be excluded. To confirm that S1P-induced cAMP generation was a receptor-mediated effect, we assessed the effect of suramin, a known inhibitor of ligand-receptor interactions (40), on the S1P-induced cAMP production. Adipocytes were preincubated with 1 mg/ml suramin for 10 min, and cAMP content was assayed with or without 30 µM S1P. As seen in Fig. 4CGo, suramin reduced S1P-stimulated cAMP generations by 60% of control, whereas the forskolin-induced cAMP increase was not affected by treatment with suramin. These results implicated the presence of a cell surface receptor that might be one of the previously reported S1P receptors or an unidentified new one responsible for activation of adenylyl cyclase by micromolar S1P.


Figure 4
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  		FIG. 4. S1P-induced cAMP generation was mimicked by Dh-S1P but not by metabolites of S1P and was attenuated by suramin pretreatment. A and B, Comparisons between S1P and its metabolites sphingosine (Sph) and ceramide (Cer) and other sphingolipids (SPC, Dh-S1P, and Pt-S1P) on cAMP generation were evaluated in adipocytes. C, Effect of suramin on forskolin- and S1P-induced cAMP production was assessed. Adipocytes were preincubated with 1 mg/ml suramin (Sura) for 10 min and then stimulated with 30 µM S1P or 3 µM forskolin (Forsk). The experiments were done three times, and each point is the mean ± SD. **, P < 0.01 vs. S1P alone.

 
Inhibitory effect of S1P on insulin-induced leptin synthesis
A previous study reported that the incubation of differentiated adipocytes with insulin induced transcription and secretion of leptin into the incubation medium (41). Leptin expression in adipocytes is directly influenced by changes in intracellular cAMP levels, with elevation of cAMP decreasing leptin expression (42). An inhibition of insulin-induced leptin production by rodent adipose tissue was reported in the presence of forskolin, a direct activator of adenylyl cyclase, or by isoproterenol, which indirectly stimulates adenylate cyclase by activating ß-adrenergic receptors (43). This evidence prompted us to investigate whether S1P can suppress insulin-induced leptin gene expression and secretion. When differentiated adipocytes were incubated with various concentrations of S1P for 18 h in the presence of insulin, leptin secretion was markedly inhibited in a concentration-dependent manner (Fig. 5AGo). The IC50 value for the inhibition of leptin secretion was about 17.8 µM for S1P. To examine whether the inhibition of leptin secretion by S1P stimulation is regulated transcriptionally, we isolated total RNA from the cells treated with S1P for 12 h in the presence of insulin and performed quantitative real-time PCR. As shown in Fig. 5BGo, insulin-induced ob gene expression was completely suppressed by treatment with S1P and ß-adrenergic receptor agonist isoproterenol. Additional experiments were performed to test whether the inhibitory effect of S1P on insulin-induced leptin synthesis is mediated by the adenylyl cyclase-cAMP pathway. As shown in Fig. 5CGo, S1P reduced insulin-induced leptin secretion by approximately 40%, and this inhibitory effect of S1P was almost reversed by 100 µM SQ22536, which is a selective adenylyl cyclase inhibitor, without significantly affecting basal level, thus indicating that S1P inhibits insulin-mediated leptin secretion through a cAMP-dependent pathway. Adiponectin is a hormone secreted by adipocytes that regulates energy homeostasis and metabolism of glucose and lipid (44). We have also tested whether adiponectin was modulated by S1P with a similar mode. However, both mRNA and protein levels of adiponectin were not changed by S1P treatment, although the level of adiponectin was dramatically reduced by forskolin treatment (data not shown).


Figure 5
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  		FIG. 5. S1P inhibits insulin-induced leptin production in a cAMP-dependent manner. A, Changes in leptin secretion were assessed by RIA. Differentiated adipocytes were incubated with 100 nM, 1 µM, 10 µM, 30 µM, and 50 µM S1P in the presence of 50 nM insulin, and the medium was harvested after 18 h for leptin measurement. B, Changes in ob gene expression were measured by real-time RT-PCR and are expressed as fold changes over vehicle-treated controls. Adipocytes were treated with 30 µM S1P or 3 µM isoproterenol (Iso) for 12 h in the presence or absence of 50 nM insulin, and RNA was analyzed for changes in ob gene expression. C, Effect of SQ22536 on S1P-induced leptin suppression. Adipocytes were incubated for 18 h with 50 µM S1P in the presence or absence of or 100 µM SQ22536. Leptin content was quantified by RIA. Results are expressed as nanograms per milliliter culture medium and represent means ± SD for three determinations. *, P < 0.05; **, P < 0.01 vs. insulin alone.

 
Effect of S1P on triacylglycerol hydrolysis in differentiated adipocytes
In adipocytes, lipolysis is another important physiological response that can be activated by cAMP-elevating signals (45). To study the effect of S1P on lipolysis, we incubated differentiated adipocytes with various concentrations of S1P for 12 h and determined glycerol level in the medium. S1P increased glycerol release in a concentration- and time-dependent manner, and the profile of S1P-induced glycerol release was well correlated with that of the cAMP increase (Fig. 6Go, A and B). The maximal glycerol accumulation in the medium was observed after 12 h incubation with 30 µM S1P. In addition, when adipocytes were pretreated with SQ22536, S1P-induced lipolysis was suppressed by approximately 48% (Fig. 6CGo), indicating that in adipocytes, S1P stimulates lipolysis through an increase in intracellular cAMP.


Figure 6
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  		FIG. 6. S1P activates lipolysis in a cAMP-dependent manner. A, Dose responses of S1P in lipolysis were evaluated in differentiated adipocytes. Adipocytes were incubated with various concentration of S1P for 24 h, and the culture media were harvested and glycerol content of the media determined and expressed as 340-nm absorbance of reduced nicotinamide adenine dinucleotide (NADH). B, Kinetics of S1P-stimulated lipolysis were assessed. Adipocytes were incubated with 50 µM S1P to stimulate lipolysis for 24 h. Aliquots of the culture media were removed at the individual times and glycerol content of the medium determined. C, Effect of SQ22536 on S1P-stimulated lipolysis. Differentiated adipocytes were stimulated with 50 µM S1P in the presence or absence of 100 µM SQ22536 (SQ) for 24 h and the concentration of glycerol in the medium determined as described in Materials and Methods. For positive controls, 3 µM forskolin (Forsk) and 3 µM isoproterenol (ISO) were used. Con, Control. Data present the mean ± SD from three experiments. *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The study in this report clearly demonstrated that the S1P-induced signaling of calcium and cAMP in cultured rat white adipocytes was distinguished in a concentration-dependent manner. Based on the above observation, we focused on clarifying the nature of the signal pathways and identifying physiological function of S1P-induced cAMP production. We found that S1P was able to stimulate lipolysis in a time- and concentration-dependent manner in white adipocytes. Moreover, the inhibition of S1P-induced lipolysis by adenylyl cyclase inhibitor SQ22536 supports the functional role of the cAMP pathway in the lipolytic process. On the other hand, we found that insulin-induced leptin secretion was suppressed by S1P-mediated cAMP signaling by quantitative real-time RT-PCR and leptin RIA. These findings illustrated physiological role of the S1P-mediated cAMP signaling in rat white adipocytes by showing that S1P has dual functions of activating lipolysis as well as suppressing leptin gene expression (Fig. 7Go).


Figure 7
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  		FIG. 7. Schematic representation showing the distinct signals of S1P between Ca2+ and cAMP and the dual roles of S1P-induced cAMP generation on lipolysis and leptin synthesis in rat white adipocytes.

 
Our results suggest that the stimulatory effect of S1P on adenylyl cyclase is caused by activation of receptors in the plasma membrane rather than an intracellular effect of sphingolipid. Although the present studies could not show direct evidence of whether the receptor coupled to adenylyl cyclase is one of the previously identified S1P receptors or an unidentified novel one, we demonstrated the existence of a receptor responsive to relatively high concentrations of S1P. First, Dh-S1P, the S1P analog that binds to all of S1P receptors with similar potency compared with S1P without mediation of intracellular action (36), effectively induced cAMP generation. Second, suramin, a general inhibitor of ligand-receptor interactions (40), reduced the stimulatory effect of S1P on cAMP generation, indicating that the sites of action for S1P are the receptors in the plasma membrane. Although in CHO cells expressing p2y9/GPR23, which was recently identified as a G-protein-coupled receptor for lysophosphatidic acid, the lysophosphatidic acid induced an increase in intracellular calcium level and stimulated adenylyl cyclase activity at a higher agonist concentration (46), a distinct concentration difference to evoke increases in cellular calcium and cAMP is not normally considered within the repertoire of cellular responses to S1P. To our knowledge, this is the first report showing a differential concentration profile between cAMP and Ca2+ increase by S1P stimulation.

Because physiological concentrations of S1P range from 0.4–1.5 µM in blood (47, 48) and S1P can be elevated to much higher levels in a local area by action of sphingosine kinase activity, we speculate that S1P-induced lipolytic activity could potentially be physiological. Some S1P receptor-mediated effects require micromolar concentrations of agonist. For example, S1P (5–20 µM) induces cell-cell aggregation in S1P receptor (S1P1)-transfected cells by up-regulation of cadherin molecules but not the mock-transfected HEK-293 cells, suggesting that the effect is receptor mediated (21). At higher concentrations (1–50 µM), S1P strongly inhibited cell attachment or cell adhesion to extracellular matrix proteins such as laminin, collagens I and IV, and fibronectin in ovarian cancer cells (49). Moreover, recent studies provide evidence that there are normally large differences in S1P availability between the circulatory fluids plasma and lymph and the lymphoid organs (50, 51). S1P abundance can be increased more than 1000-fold in the thymus, lymph node, and Peyer’s patch in certain conditions such as inhibition of S1P lyase activity (47). These data raise the possibility that endogenous S1P receptors may have potential to respond to micromolar S1P. Because sphingosine kinase is strongly activated in the signaling pathway of acute inflammation (52, 53, 54), it is plausible speculation that the effects of micromolar S1P is related to a pathophysiological condition rather than a physiological condition. Inflammation accompanied by cytokine release (52), complement activation (54), or thrombosis (53) may promote the metabolism of ceramide to generate S1P via sphingosine in white adipose tissues. This condition may permit S1P-mediated down-regulation of leptin synthesis and activation of triacylglyceride hydrolysis through autocrine or paracrine mechanisms.

Our work reveals that S1P signaling in white adipocytes has dual effects that are inhibitory in leptin synthesis and stimulatory in lipolysis. In addition to S1P, some agonists (TNF-{alpha} and ATP) that have inhibitory effects on insulin-mediated leptin synthesis also have stimulatory effects on lipolysis in adipocytes (12, 55, 56). A wide range of agents known to increase intracellular cAMP levels by stimulating adenylyl cyclase or by inhibiting phosphodiesterases suppressed insulin-induced leptin secretion and concomitantly stimulated lipolysis and fatty acid release (45). However, it remains to be elucidated how they can be coupled in adipocytes and what role they play to link lipolysis and leptin production. Although we demonstrated and characterized for the first time that S1P in rat white adipocytes leads to an inhibition of insulin-mediated leptin synthesis and activation of lipolysis in a cAMP-dependent pathway, the identification of the S1P receptor responsible for cAMP generation is crucial to further elucidate the precise roles of S1P in the regulation of leptin synthesis and lipolysis.


   Footnotes
 
This work was supported by the Brain Neurobiology Research Program Grant M10412000088-04N1200-08810 and Systems Bio-Dynamics National Core Research Center sponsored by the Korean Ministry of Science and Technology and Brain Korea 21 Program of the Korean Ministry of Education.

Disclosure statement: The authors have nothing to disclose.

First Published Online September 14, 2006

Abbreviations: CREB, cAMP response element-binding protein; Dh-S1P, dihydro-S1P; EDG, endothelial cell differentiation gene; IP3, inositol-1,4,5-trisphosphate; JNK, c-Jun N-terminal kinase; PLC, phospholipase C; Pt-S1P, phyto-S1P; PTX, pertussis toxin; S1P, sphingosine-1-phosphate; SPC, sphingosylphosphoryl-choline.

Received May 2, 2006.

Accepted for publication September 6, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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